3D KIDNEY ORGANOIDS AS A PLATFORM FOR INVESTIGATING MONOGENIC DISEASES AND GENE THERAPY APPROACHES

Abstract The use of 3D in vitro models is now widely adopted in biomedical research. These models enable the study of human cells under more physiologically relevant conditions and allow for early proof-of-concept testing of therapies prior to animal studies. While 3D models do not replace traditional 2D or animal models, they represent a powerful complement. Here, we used human induced pluripotent stem cells (iPSCs) to generate kidney organoids as a 3D model of rare inherited kidney diseases. Human kidney organoids are an invaluable platform for studying renal pathophysiology, developing cell-based therapies, and testing novel therapeutic approaches. We generated and characterised kidney organoid models of proximal tubulopathies such as Lowe syndrome, Fabry disease, and Cystinosis. These were characterised at the morphological and functional level, displaying pathology-relevant phenotypes useful for validating therapies such as Enzyme Replacement Therapy (ERT) and Substrate Reduction Therapy (SRT) for Fabry disease and pharmacological therapy for Lowe syndrome. However, despite being widely adopted in clinics, these therapies have proven to be only partially effective, indicating that new therapeutic strategies are needed. Looking around, across the global research landscape, there has been a remarkable acceleration in the advancement of AAV-based gene therapies, largely due to accumulating evidence supporting their safety and therapeutic efficacy. Consequently, this has become one of the major focuses of current biomedical research. Given the limited knowledge and tools to target specific cells within the kidney, we used kidney organoids to screen 12 different AAV vectors. Our screening demonstrated that the transduction efficiencies in both wild-type and pathological conditions were comparable and that not all the serotypes showed the same cellular tropism; notably, mainly AAV2/2, AAV2/3 and AAV7m8 showed a strong specificity for human proximal tubular cells within human iPSC-derived kidney organoids. We have selected AAV2/2 as the master serotype for our upcoming proof-of-concept studies. The ultimate aim is to provide a proof-of-concept of the efficacy of a novel AAV-based gene therapy in Lowe syndrome, which we selected as a prototype of inherited tubulopathy. We have previously demonstrated, using various pharmacological compounds, that kidney organoids are a reproducible and reliable in vitro model to assess that purpose. Indeed, gene therapy efficacy can be substantiated not only by assessing static phenotypic markers such as intracellular substrate accumulation, autophagy dysregulation and other related cellular alterations, which we evaluate through immunofluorescence and Western blotting, but also by incorporating functional assays. In particular, we have established an endocytosis assay to assess proximal tubule function in our kidney organoid model. Through this multifaceted approach, we aim to demonstrate the therapeutic potential of AAV-mediated gene delivery in restoring proximal tubule physiology.